Indole-3-carbinol (I3C), an indolecarbinol compound obtained from Brassica vegetables, such as broccoli, cabbage and Brussels sprouts, exhibits potent anti-proliferative properties in a wide range of human cancers with negligible levels of toxicity or side effects. We have documented that I3C, and its more potent and stable derivative 1-benzyl-I3C, triggers complementary sets of transcriptional, cell signaling, and enzymatic cascades that control cancer cell growth, apoptosis, cell migration and in vivo tumor growth of human cancer cells. Our studies originally established the serine protease elastase as the first known I3C target protein in human reproductive cancer cells, which has provided a crucial experimental foundation to define the mechanisms of indolecarbinol anti-proliferative signaling in other cancer cell types. In human melanoma cells, we have observed that I3C induces a G1 cell cycle arrest and inhibits cell survival signaling by stabilizing the PTEN tumor suppressor protein levels, which through its dual lipid/protein phosphatase activity then disrupts the Akt signaling network including the inactivation of NFkB transcriptional activity and attenuated expression of NFkB target genes such as cyclin D1. I3C triggers this response by inhibiting the NEDD4-1 mediated ubiquitination of PTEN, and in silico 3-D simulations using the crystallographic structure NEDD4-1 predicts that I3C specificity interacts with the HECT domain of NEDD4-1, which is the domain responsible for the E3 ubiquitin ligase activity. This proposal will test the hypothesis that that the NEDD4-1 E3 ubiquitin ligase, which selectively ubiquitinates and signals the 26S proteasome degradation of PTEN suppressor protein, is directly inhibited by I3C and represents a new indolecarbinol target protein that mediates I3C anti-proliferative responsiveness of human melanoma cells. One aim of this proposal will utilize in vitro ubiquitination, I3C binding and protein (or domain) interaction assays to determine the mechanism by which I3C inhibits the NEDD4-1 E3 ubiquitin ligase activity. A mutagenic strategy will alter the in silico defined I3C interaction site to define the precise amino acid requirements for I3C binding and generate novel I3C-resistant forms of NEDD4-1. The second aim will functionally characterize I3C activated anti-proliferative and pro-apoptotic cascades due to PTEN protein stabilization in human melanomas cells with distinct phenotypes. Also, more potent and selective derivatives of I3C will be identified based on their interactions with NEDD4-1 and by their ability to stabilize PTEN protein and disrupt Akt/NFkB signaling. The third aim will characterize the anti-cancer effects in melanoma cells of combinations of indolecarbinol compounds and aspirin in both cellular and in vivo contexts. Our proposed studies will establish the preclinical foundation that is needed to eventually develop novel and low cost I3C-based therapeutic strategies for human skin cancers.